Trisomy 21, commonly known as Downs Syndrome, is characterised by an extra copy of chromosome 21 and is one of the most common serious congenital abnormalities resulting in the most frequent single cause of significant learning disability in children of school age. People afflicted with Downs Syndrome have severe mental retardation, reduced life expectancy and abnormal immune response that predisposes them to serious infections. 40% of Downs Syndrome sufferers have congenital heart disease and an increased risk of developing leukaemia. All people over 40 with Down's Syndrome are liable to develop neuropathological changes characteristic of Alzheimer's disease.
The definitive test for Down's Syndrome in early pregnancy, i.e. at about 15 to 16 weeks, is karyotyping following amniocentesis. The sampling of amniotic fluid required for this carries the risk of inducing spontaneous miscarriage, which may occur in about 1 in 100 cases.
Originally maternal age was the only factor used to identify women at high risk of having a Downs Syndrome baby. At age 40 the chance of having a Downs baby is 1 in 100. This has led to many hospitals offering amniocentesis to women over a certain age, usually 35 or 37. However, this will only identify 15-30% of all cases of Down's syndrome as the majority still occur in women who are younger.
Over the past 25 years multiple marker blood tests have been developed to screen for Downs Syndrome. Serum alpha-fetoprotein (AFP) plus human chorionic gonadotrophin (hCG) are the most common markers. More recently, however, unconjugated estriol (uE3) and inhibin A have been added to the markers screened for.
Screening performance varies according to the choice of markers used and whether ultrasound is used to estimate gestational age and to measure nuchal fold thickness at 15-22 weeks. When screening for serum markers and ultrasound are used in combination with maternal age, the detection rate for a 5% false-positive rate is estimated to be 59% for the double test (AFP+hCG), 69% for the triple test (AFP, hCG, uE3) and 76% for the quadruple test which includes inhibin A.
Current screening technology is expensive and requires the minimal invasive procedure of blood sampling together with mathematical modelling of values detected corrected against level changes due to gestation age.
Human chorionic gonadotropin (hCG) is a glycopeptide hormone produced by the syncytiotrophoblasts of the fetal placenta, and has a molecular weight of about 38 kilodaltons. It can be detected by immunoassay in the maternal urine within days after fertilisation. The intact hCG molecule is a heterodimer comprising a specific β 25 subunit non-covalently bound to an a subunit, which is common to other glycoproteins.
Maternal serum levels of both intact hCG and the free β-subunit are elevated on average in Down's Syndrome, but the extent of elevation is greater for 30 free β-hCG. HCG is detected in both the serum and urine of pregnant women, as are the free α and β subunits of hCG, as well as the degradation products of hCG and of free β-subunit hCG.
The terminal degradation product of the β-subunit of hCG is urinary gonadotropin peptide (UGP), otherwise known as β-core-hCG, β-core fragment, β-core or urinary gonadotrophin fragment (uGF).
UGP is excreted into urine. WO97/03363 describes a pre-natal urinary screening method for Down's Syndrome which comprises testing a maternal urine sample during the first trimester of pregnancy to determine whether the level of UGP in the sample is elevated above the level of UGP found in urine samples from normal 10 pregnancies. The method described in WO 97/03363 utilises immunoassays specific for UGP.
The precise structure of hCG has been well characterised by HPLC-mass spectrometry and by crystallographic analysis. Using trypsin digestion, peptide mass mapping of hCG and its subunits has been carried out using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS). Jacoby, E. S. et al in Clinical Chemistry, 46 (11), 1796-1803 (2000) described the purification from pregnancy urine of hCGβ-Core fragment (hCGβcf), which was reduced by dithiothreitol (DTT) reduction and analysed by MALDI-ToF MS. They concluded that the mass spectrum of DTT-reduced hCGβcf that was produced, whilst not precisely defining hCG β cf glycosylation, would appear to result in a distinctive “fingerprint”. MALDI-ToF MS analysis of unextracted urine samples from pregnant women showed only a broad peak corresponding to proteinated hCGβcf.
WO03/065043 describes the immobilisation of immunoglobins on a MALDI-TOF MS target which may then be used in the identification and relative quantification of proteins such as hCGβcf.
Human chorionic gonadotropin (hCG) is a hetro-dimeric glycoprotein hormone with 8 glycosylation sites containing four N-linked oligosaccharides and four O-linked oligosaccharides. The N-linked oligosaccharides are attached to the polypeptide chain by β-N-glycosidic bonds on asparagine residues; two are on the α and two are on the β-subunit. They share the same basic structural characteristics: N-acetyl glucososamine (GLcNAc) is attached to an asparagine residue followed by another GLcNAc, mannose and two more branches of mannose. This is the monantennary pentasaccharide core with the remaining components being variable. The O-linked oligosaccharides are attached by α-β-glycosidic bonds onto serine residues of the β-subunit carboxyl terminal peptide.
Carbohydrate heterogeneity has been extensively reported for the free β-subunit of hCG (hCG β) with variable mono-, bi- and triantennary carbohydrate structures being found in normal and abnormal pregnancies (Elliott M M, Kardana A, Lustbader J W, Cole L A. Endocrine. 1997 August; 7(1):15-32. Carbohydrate and peptide structure of the alpha- and beta-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma).
The degradation product of the β-subunit of hCG known as β-core fragment (hCG β cf) is composed of peptides from the β-subunit of hCG, i.e peptides β 6-40 and β 55-92, connected by four disulfide bridges. It retains many of the antigenic determinates of the original hCG β molecule prior to metabolism, which occurs primarily in the kidney. The β 6-40 polypeptide chain contains the two hCG β N-linked carbohydrate moieties, although the oligosaccharides are truncated due to metabolism.
Whilst hCG β cf glycosylation has been studied using MALDI-ToF MS, which resulted in a mass spectrum that appeared to give a distinctive “finger print”, the method used involved a pre-treatment of samples with dithiothreitol to reduce the mass of peptides thus bringing them into a relatively optimum resolution range of the mass spectrometer. MALDI-ToF MS analysis, according to Jacoby et al (2000) resulted in a broad peak corresponding to protonated hCG β cf.
The disadvantage of MALDI-ToF MS is that it is not a quantitative technique. Consequently, the raw mass spectra produced cannot be used directly to produce comparative data for diagnostic test algorithms.